Use of compositions comprising stabilized biologically effective compounds

ABSTRACT

A dual chambered dispensing system allows for application of an aqueous composition containing a biologically effective compound which is adequately stabilized. The system separately contains the stabilized biologically effective compound composition in one chamber and an aqueous basic composition in the other. Both compositions are simultaneously delivered from the dispensing system, whereupon the compositions are mixed to result in a final composition suitable for direct application.

This application is a national stage filing under 35 USC § 371 ofinternational application No. PCT/EP97/00507, filed on Jan. 31, 1997.

FIELD OF THE INVENTION

The present invention relates to the field of application ofcompositions comprising stabilized biologically effective compounds byuse of a multicomponent dispensing system.

BACKGROUND OF THE INVENTION

Topical application of enzymes has been described in the cosmetic aswell as in the pharmaceutical field. For example, the use of proteaseshas been suggested to support or replace α-hydroxy acids in skin peelingpreparations (Japanese patent application J04027388). Glutathionsulfhydryl oxidase has been identified as useful in hair-wave setting(Japanese patent application J04005220). Furthermore, Internationalpatent application WO93/19731 describes the use of glycosidases toenhance the process of skin desquamation and lysozyme has been mentionedto treat acne (HUT 057608). More recently, several patent applicationsusing the enzyme transglutaminase have been published (WO94/18945,J02204407).

However, the limited storage stability of enzymes in liquid aqueousformulations is to be considered as the main limiting factor to thewider application of enzymes.

Commercial preparations containing enzymes often take advantage of theshelf stability of an enzyme in a dry state. According to such aconcept, the easiest way to market an enzyme-containing product is bysupplying the enzyme separately with the product, e.g. suitably packedas tablets. In an alternative approach, dry enzyme powder can behomogeneously dispersed into an essentially non-aqueous hydrophobicbase, such as a suitable oil in combination with an oil gellant.

Disadvantage of the first approach is that the required dissolution ofthe enzyme tablet in an aqueous composition is slow and inconvenient.Regarding the second approach it should be noted that an enzyme requireswater to be active. In order to be efficient, the mixing of an aqueousand an oily phase generally requires a relatively high energy input andcannot be achieved by simple hand-mixing. Thus, the mixing of an aqueouscomposition and a hydrophobic enzyme-containing phase is expected to bevery inefficient.

The above problems can be circumvented by using aqueous stabilizedenzyme compositions for topical applications. Unfortunately, aqueousenzyme formulations require high concentrations of water-misciblestabilizers intended to lower the water activity of the formulation.Polyols are often used for this purpose and long-term stability can onlybe effected by polyol concentrations well above 40% (v/v). However,enzymes often are not active in compositions containing high polyolconcentrations. Especially the direct topical application ofthus-stabilized enzyme composition will not provide sufficient water toreactivate the enzyme. Moreover, the presence of such high polyolconcentrations in compositions for topical use is consideredunacceptable.

Consequently, the high polyol concentration which is necessary tostabilize an enzyme in an aqueous environment prevents direct topicalapplication of a thus-stabilized aqueous enzyme composition.

Another area in which enzymes can be advantageously used is the area oflaundry hand wash applications. Although compared to machine washing theincidence of hand washing is very low in Europe and North America, handwashing remains popular as far as delicate fabrics are concerned. Of thedelicate fabrics, the small category of woollen and silk itemsrepresents a particularly problematic area regarding stain removal,fabric depilling, colour revival and fabric shrinking. This category ofspecific fabrics may require specific enzymes, such as proteases activearound a neutral pH value and/or at a low temperature or sulphur bridgerearranging enzymes like protein disulfide isomerase to counteract wooldeformation stresses exerted during washing (EP 276547). A disadvantageof such niche products is that they obviously cannot bear the cost ofextensive detergent or enzyme formulation developments.

Similarly, various biologically effective compounds other than enzymesare known which are unstable in aqueous end formulations, i.e. thoseformulations which are suitable for direct use in a specificapplication. Typically, biologically effective compounds like enzymes,antibiotics, vitamins, polyunsaturated compounds and the like, loosetheir activity upon prolonged storage in aqueous compositions. Althoughspecific formulations are known in which said biologically effectivecompounds are stably incorporated, the latter formulations typically arenot suitable for direct use in desired applications.

SUMMARY OF THE INVENTION

The present invention discloses a dispensing system separatelycontaining a first and a second aqueous composition, said firstcomposition being a composition comprising a biologically effectivecompound which is stably formulated, wherein said first and secondcomposition generate a final composition when mixed upon dispensing, andwherein said final composition is effective to apply said biologicallyeffective compound in an active form.

In the dispensing system of the invention, an aqueous compositioncomprising a stabilized biologically effective compound and an aqueousbasic composition are separately contained. Using the dispensing system,simultaneous delivery of both aqueous compositions is allowed. Upondelivery, both compositions are mixed, resulting in a dilution of thecomposition comprising the stabilized biologically effective compound inthe basic composition, generating a final composition suitable fordirect application.

Preferred biologically effective compounds for use in the dispensingsystem of the present invention are enzymes, vitamins, polyene macrolideantibiotics, dihydroxyacetone, and aldehyde flavours. The dispensingsystem of the present invention is especially suitable for topicalapplication of a biologically effective compound.

DETAILED DESCRIPTION OF THE INVENTION

In the dispensing system of the present invention, an aqueouscomposition comprising a stably formulated biologically effectivecompound and an aqueous basic composition are separately contained.

Using said dispensing system, simultaneous delivery of the aqueouscomposition comprising a stably formulated biologically effectivecompound (called the "effective composition" or "first composition"throughout the invention) and the aqueous basic composition (also calledthe "second composition") is possible. Upon delivery, both aqueouscompositions are mixed, either in situ or in the dispensing system.Mixing of both compositions results in a final composition whichcontains a biologically effective compound in an active form and whichadditionally is suitable for direct use.

The term "basic composition" is used for a composition which produces incombination with the aqueous composition comprising the biologicallyeffective compound a final composition which is suitable for directapplication of the biologically effective compound. The nature of thebasic composition mainly will depend on the desired application. Aqueousbasic compositions are understood to include oil-in-water emulsions.

Preferably, the aqueous basic composition is a composition suitable fortopical, detergent or cleaning use. More preferably, the aqueous basiccomposition is a composition suitable for topical use. Most preferably,the aqueous basic composition is a composition suitable for cosmeticuse.

The aqueous basic composition can be a cream, a gel, a shampoo, acleansing fluid, a lotion, a liquid detergent, a hard surface cleaningcomposition, and the like.

Biologically effective compounds which are suitable for use in thedispenser of the present invention are those compounds which display abiological activity and which are unstable in the aqueous endformulation in which the biologically effective compound should beapplied. In addition, biologically effective compounds which aresuitable for use in the dispenser of the present invention are thosecompounds for which a stable aqueous formulation is developed, whichstable formulation is not suitable for direct use in the desiredapplication.

The biologically effective compounds which are suitable for use in thedispenser of the present invention are distinguishable with respect tothe source from which said compound is obtainable as well as withrespect to the nature of said compound.

With respect to the source of the biologically effective compound, saidcompound is obtainable from an animal, a vegetable or a microbialsource. Preferably, said compound is obtainable from a microbial or avegetable source. More preferably, said compound is obtainable from amicrobial source.

With respect to the nature of the biologically effective compound, saidcompound is selected from the group of primary and secondarymetabolites, preferably from the group of enzymes, antibiotics,(poly)unsaturated compounds, vitamins, flavours, dihydroxyacetone, morepreferably from the group of enzymes, vitamines, polyene macrolideantibiotics, aldehyde flavour compounds and dihydroxyacetone.

The instability of a biologically effective compound in an aqueousenvironment, which is particularly perceptible after prolonged storage,may be of a chemical nature, for instance caused by structuraldeterioration (e.g. denaturation in case of enzymes and other proteins),oxidative attack or other unfavorable conditions like non-optimal pHconditions. Oxygen, as well as light and the presence of metal ions fromtraces of iron or copper, are known for their detrimental oxidativeeffect on biologically effective compounds like vitamins, carotenoids,(poly)unsaturated oils and (poly)unsaturated fatty acids (see forexample CRC Handbook of Food Additives, second edition). Instability mayadditionally be caused by microbial growth in an aqueous environment, orby physical instability of the aqueous composition containing thebiologically effective compound.

Depending on the factor(s) causing instability of the biologicallyeffective compound, stable aqueous formulations were developed which arecharacterized by, for instance, one or more of the following conditions:a low water activity, a low or a high pH, a high concentration of anantioxidant, a high concentration of a sequestering agent, a highconcentration of an antimicrobial agent, crystallinity of thebiologically effective compound, a high concentration of a viscosifyingagent. Typically, said condition(s) necessary for stabilizing thebiologically effective compound in an aqueous composition do(es) notallow the direct use of said aqueous composition.

The dispensing system of the invention enables the use of relativelyhigh concentrations of chemical stabilizers to prepare stableformulations of inherently unstable biologically effective compounds,i.e. concentrations which can be much higher than those allowed in afinal composition, since the stabilizers are diluted with an aqueousbasic composition upon dispensing. Suitable stabilizing agents includewater activity lowering agents like salts or polyols, sequesteringagents like EDTA, phytate or gluconate or antioxidants like sulphites,glutathion, cysteine or ascorbic acid.

In another aspect of the invention, the use of the dispensing systemensures that the effective concentration of a biologically effectivecompound is reached after dilution of the effective composition in thebasic composition. Therefore, the biologically effective compound can bepresent in the effective composition in a considerably higherconcentration than would be required for efficacy. Several biologicallyeffective compounds are insoluble in aqueous compositions in thesehigher concentrations. This implicates that the biologically effectivecompound can be present in a crystalline form. Said crystalline form isespecially advantageous to ensure stability of the compound.

However, an important problem with compositions comprising crystallinecompounds is that crystals tend to sediment in such compositions, i.e.said compositions are physically unstable.

The present invention discloses that sedimentation of a crystallinecompound is prevented by the use of a suitable viscosifying agent. Saidviscosifying agent preferably is able to form a three-dimensionalnetwork in an aqueous environment. More preferably, said viscosifyingagent is selected from the group of xanthan, Carbopol® or relatedresins, or carrageenan. Most preferably, said viscosifying agent isxanthan. The concentration of a suitable viscosifying agent mainly isdetermined by the weight and size of the particles to be kept insuspension. Conveniently, the concentration may range from 0.1-3%,preferably from 0.2-0.6%.

The dispensing system of the present invention allows for the dilutionof the stabilizing agent present in the composition containing abiologically effective compound upon dispensing with the basiccomposition. The dispensing system of the invention further allows forthe dilution of the effective compound to its effective concentration.

The dilution factor of the composition containing a biologicallyeffective compound (the effective composition) in the basic compositionis adequately chosen, i.e. such that the end concentration of thestabilizing agent does not preclude application of the final compositionand such that the biologically effective compound is present in thefinal composition in its appropriate effective concentration. Thedilution factor is determined by the ratio in which the effectivecomposition and the basic composition are delivered by the dispensingsystem. Preferably, the ratio between the effective composition and thebasic composition varies from 1:1 to 1:50, more preferably from 1:2 to1:20, most preferably the ratio is 1:5 to 1:10.

According to the invention, the viscosity of the effective compositionpreferably has a value which is comparable to the viscosity of the basiccomposition which is applied simultaneously with the compositioncontaining the biologically effective compound. For instance, bothcompositions can have a lotion-like, a cream-like or a gel-likeconsistency. The viscosity of the effective composition additionallywill depend on the type of dispensing system which is used to deliverthe compositions. For instance, the use of a tube requires a relativelyhigh viscosity of both compositions.

The amount of viscosifying agent to be added to the effectivecomposition will depend among others on the desired viscosity of saidcomposition. Any viscosifying agent known to the skilled person which iscompatible with the final composition as well as the desired applicationcan be used. For instance, for topical application its acceptability fortopical use should be considered. Examples of viscosifying agentsinclude carrageenans, cellulose derivatives, polyacrylic acids, clays,polyethylene glycols, hydrocolloids such as xanthan.

If desired, agents can be added to the effective composition and/or thebasic composition, such that both composition have the same or have adifferent appearance. A typical example of such an agent is a colourant.

The aqueous effective composition is understood to include oil-in-wateremulsions.

To obtain a desired shelf stability of compositions containingbiologically effective compounds which are prone to oxidation,non-translucent packaging material with a low ingress of oxygen would bedesirable. The dispensing system of the present invention provides theoption to pack the composition containing the biologically effecticecompound in a compartment made from non-translucent material with verylow oxygen permeation rates even under conditions of high humidity, tominimize the effect of light and ingress of oxygen. Preferred packagingmaterials include PVdC, EVOH and alumina-coated polymers (see FoodManufacture, June 1991, pp 49-53). If applied in larger volumedispensers, the use of an air-free lotion pump to dispense thebiologically effective compound is another requirement.

The dispensing sytem to be used in the method of the invention is notcritical to the invention. The present invention contemplates any systemwhich allows for the separate containment of the stabilized effectivecomposition and the basic composition. Separate containment isunderstood to include any form of separation which is able to prevent asubstantial diffusion of water from one to the other composition.

For instance, a dispensing system can be selected from themulticomponent dispensing systems which have been developed for thepackaging and delivery of non-compatible chemical compounds, i.e.chemical compounds which react with each other when brought intocontact. For instance, multicomponent dispensers are known from thefield of adhesives. The packaging of multicomponent adhesives requirescomplete separation of the resin and the hardener. Yet, convenience inuse requires simultaneous delivery of the two components.

Apart from adhesives, multicomponent dispensing systems have also beendescribed for the formulation of incompatible compounds in toothpaste.Flexible two-compartment dentrifice tubes are described in U.S. Pat.Nos. 4,487,757, 4,098,435 and U.S. 4,211,341. The latter patentdiscloses the use of extrudable materials such as carboxymethylcellulosegel in a polyhydric alcohol solution to separate the incompatiblecompounds. A two-compartment tube for the storage of a non-aqueousenzyme composition separate from the aqueous toothpaste composition hasbeen described in FR 2,051,922.

In yet another but basically very simple approach, one pair of plasticpouches provides material for single use only. The outlets of the twopouches are close to each other and discharge of the contents can beeffectuated by tearing open the end pieces of the pouch (German patentapplication DE 3 630 849).

The present invention also contemplates the formulation and storage ofthe stabilized effective composition and the basic composition inseparate containers, which are put together and/or provided with asuitable dispenser by the user. It is also possible that a dispensingsystem already provided with a container containing one composition,e.g. the stabilized effective composition, is additionally provided witha container with the other composition, e.g. the basic composition.

The dispensing system of the present invention can be conveniently usedfor any application wherein the action of a labile biologicallyeffective compound is desired. In particular, the dispensing system ofthe invention provides a convenient and simple way for topicalapplication of a biologically effective compound of interest. Topicalapplication is understood to include application on skin and hair andapplication in the oral cavity, e.g. on teeth.

The applicability of the dispensing system of the present invention isnow illustrated in the light of several biologically effectivecompounds.

Enzymes

Typically, an aqueous enzyme composition is stabilized with a highconcentration of a water activity lowering agent, such as a polyol or asalt. Preferably, a polyol is used for stabilization.

Using the dispenser of the present invention, the mixing of the enzymeand the basic composition results in an actual dilution of the enzymecomposition in the basic composition. The high concentration of forinstance a polyol in the enzyme composition guarantees activity of anenzyme upon dilution, even after a prolonged storage period of theenzyme composition.

Said dilution of the enzyme composition results in a dilution of thepolyol, which on its turn results in a reactivation of the enzyme.Depending on the enzyme and the polyol used, enzyme reactivation can beexpected to start at polyol concentrations below 40% w/w.

The ratio in which the enzyme composition and the basic composition aredelivered by the dispensing system depends for instance on theconcentration of the polyol in the enzyme composition, whereby the ratioshould be adjusted in such a way to ensure reactivation of the enzyme.Furthermore, if topical use is desired, said ratio should be adjusted insuch a way that the concentration level of the polyol, after mixing theenzyme with the basic composition, does not exceed the acceptable levelfor use in topical formulations.

Use of the dispensing system of the invention enables the stableformulation and application of any enzyme of interest. Preferably, theenzyme of interest belongs to the class of oxidoreductases,transferases, hydrolases or isomerases. More preferably, the enzyme is aglucose oxidase, peroxidase, lipoxygenase, superoxide dismutase,tyrosinase, protease, phosphatase, phytase, glycosidase, glucanase,mutanase (α-1,3-glucanase), dextranase, lysozyme, lipase, phospholipase,sulfatase, urease, transglutaminase or protein disulfide isomerase. Itis also possible to apply a stabilized composition comprising a mixtureof two or more enzymes.

The concentration of the enzyme in the enzyme composition mainly will bedetermined by the type of application.

The present invention also envisages enzyme compositions in which theenzyme is formulated in a particle form. Enzymes formulated asparticulate matter greatly reduce the risk of sensitization which mayoccur upon potential inhalation of enzyme molecules when dried afterapplication. Preferably, the enzyme is formulated as particles having aparticle size of at least about 5-10 μm. The upper limit of the particlesize of the enzyme particles generally will be determined by the factthat larger particles will have an unfavourable surface loading and mayproduce a gritty feeling upon application to the skin. Conveniently, theupper limit of the particle size is about 100 μm.

One method to obtain enzyme particles of at least about 5-10 μm is tocovalently immobilize the enzyme on a suitable carrier, as described ine.g. Methods in Enzymology, vol. 44 (1976). Another example of asuitable particle form is a so-called ChiroCLEC (Altus Biologics Inc.,Cambridge, Mass., USA), which consists of cross-linked enzyme crystals.These cross-linked enzyme particles do not need the presence of highconcentrations of a water activity lowering agent such as a polyol forstable formulation; they are chemically stable in an aqueous compositionbecause of their crystalline form. Nevertheless, water activity loweringagents may still be added to improve microbiological stability of theaqueous composition.

The choice of the polyol which is used to stabilize the enzymecomposition is not critical for the invention. Any polyol which is knownto the skilled person to effectively stabilize enzymes in aqueoussolutions can be used. Polyols that are particularly useful are polyolsselected from the group of glycerol, sorbitol, propylene glycol,maltodextrins, or a sugar such as sucrose, lactose, glucose ortrehalose. For topical applications, one should consider a polyol whichis acceptable for topical use, i.e. glycerol, polyethylene glycol,butylene glycol, propylene glycol, trehalose or sorbitol.

The polyol is used in a high concentration, i.e. a concentration whichresults in a sufficiently low water activity in the enzyme compositionto adequately stabilize the enzyme. It is known in the art that theseconcentrations may somewhat vary with the polyol used. Preferably, thepolyol is used in a concentration of 20-90%, more preferably in aconcentration of 30-90%, even more preferably in a concentration of40-90%, even more preferably in a concentration of 50-90%, mostpreferably in a concentration of 60-80%.

A low water activity in an aqueous composition is also advantageous forpreventing microbial growth in the composition.

In addition to a polyol, a salt such as NaCl may be used to enhance thestability of the enzyme during the product's shelf life. To furtherimprove enzyme stability, low concentrations of enzyme stabilizers suchas reducing agents, calcium salts or substrate or substrate-relatedligands may be added (Gray, 1993, in: Thermostab. Enzymes, pp. 124-143.Narosa, New Delhi).

Optionally, a viscosifying agent may be added to the enzyme composition,in particular if the viscosity of the enzyme composition due to thepolyol or other relevant components is not as high as is desirable. Theamount of viscosifying agent to be added to the enzyme composition willdepend on the viscosifying properties of the polyol which is used forstabilization of the enzyme composition as well as on the desiredviscosity of the enzyme composition.

If an immobilized or crystalline enzyme preparation is used, theviscosity of the enzyme composition should be such that sedimentation ofenzyme particles is prevented. Preferably, as is indicated before, aviscosifying agent is used which is able to form a three-dimensionalnetwork in an aqueous composition.

According to the invention, the enzyme composition will be essentiallysimultaneously delivered with an appropriate aqueous basic composition.The nature of this aqueous basic composition is not critical for theinvention, but will mainly depend on the type of application which isdesired.

Attention should further be paid to avoid that the aqueous basiccomposition contains components that can be expected to inactivateenzymes instantaneously For cosmetic lotions, a typical example of acomponent expected to inactivate enzymes is ethanol in highconcentrations.

The dispensing system of the present invention can be conveniently usedfor any application wherein the action of an enzyme is desired. Inparticular, the dispensing system of the invention provides a convenientand simple way for topical application of an enzyme of interest. Apreferred enzyme for topical use of the dispenser of the invention is aprotease.

The dispensing system of the invention is also suitable tosimultaneously deliver an enzyme composition and a second compositioncomprising a proactive substrate, whereby the enzyme converts theproactive substrate into an active ingredient upon delivery and mixingof both compositions. This embodiment of the invention is preferred whenthe active ingredient is unstable in a particular composition and thepossibility exists to formulate a precursor of the active ingredient,the so-called proactive substrate, which is more stable.

For instance, vitamin E-acetate, vitamin A-acetate and vitaminA-palmitate represent precursor molecules typically used to apply theseunstable but desirable vitamins on the skin. Due to enzymatic activityin or on the skin, part of the precursor is believed to be slowlyconverted into the active compound (see for example: Boehnlein et al.,Pharmaceutical Research Vol. II, no. 8 (1994), 1155-1159). Following theenzyme dispensing method of the invention, combining such shelf stableprecursors and the appropriate hydrolytic enzymes, active retinol ortocopherol could be released on the skin. An important advantage of theuse of the dispensing system of the invention is that the hydrolysisrate of the precursor molecules significantly is increased as comparedto the situation where one depends on relevant enzymes which are presentin the skin. In certain applications, e.g. in anti-sunburn applications,the benefits of an instantaneous release of the desired concentrationsof vitamin A are evident (see for example Beijersbergen van Henegouwenet al, Fat Sc. Technol. 94 (1992), 24-27).

To activate palmitate derivatives of either vitamin A or vitamin E, theuse of a suitable lipase is an obvious choice. It can be expected thatmany of the commercially available lipolytic enzymes will be able tohydrolyse these precursor molecules into the active vitamin and palmiticacid. However, the use of a lipase in cosmetic applications has someserious disadvantages including the breakdown of oils present incosmetic compositions and the degradation of a considerable portion ofthe protective lipid compounds present on the human skin (Cosmetics &Toiletries 102 (1987), 36-42). To avoid this undesirable situation it isadvantageous to use acetate rather than palmitate derivatives of therespective vitamins, enabling the use of enzymes which are capable toselectively remove the acetate moiety of the vitamin precursor withoutattacking skin lipids.

For example, certain esterases/lipases have a preference for short-chainacyl groups (from 2-10 carbon atoms) and are not capable to hydrolyzelonger (≧16 carbon atoms) fatty acyl groups. Such esterases arecommercially available from for instance Recombinant Biocatalysis Inc.(Philadelphia, USA). In addition, xylan acetylesterase (cf. EP 507369)and rhamnogalacturan acetylesterase (cf. WO 93/20190) are active towardsplant cell wall components and unlikely to hydrolyse lipids present onthe human skin. Apart from this category of enzymes, esterase activityhas also been attributed to certain serine proteases. The use of asuitable serine protease in this application is advantageous because itallows the combination of a skin peeling effect with the simultaneousconversion of a selected vitamin precursor.

Another vitamin, ascorbic acid, has claimed benefits in cosmeticsbecause of its inhibitory effect on melanin formation in human skin, itsstimulation of collagen formation and its antioxidant activity.Unfortunately, ascorbic acid cannot be applied to any cosmetic productbecause of its poor stability. Therefore, magnesium ascorbyl phosphate,a stable and water-soluble ascorbic acid derivative, has been developedand commercialised by several companies. Due to the presence offosfatase enzymes on the skin, magnesium ascorbyl phosphate can beconverted in the active but unstable ascorbic acid in situ.Unfortunately, the activity of fosfatase enzymes which naturally occuron the skin is rather low (Mima et al. Vitamins 41 (1970), 387). Thedispensing system of the invention enables the combination of ascorbylphosphate and a suitable phosphatase to ensure rapid formation ofascorbic acid on the skin. The enzyme phytase, catalyzing the release ofphosphate from inositol-hexakisphosphate (phytate), in particular thephytase from Aspergillus niger, appears to be a very suitablephosphatase in this regard.

Very similar to the approach in which proactive vitamin derivatives areactivated by the in situ removal of the stabilizing moiety of thederivative, other types of precursor molecules can be enzymaticallymodified. Several glycosylated natural colourants are known includinganthocyanins and the food grade carmine red. As described by Blom (FoodChemistry 12 (1983), 197-204), the combination of β-glucosidase and redanthocyanin pigment results in a water-insoluble, coloured aglycon.Using the dispensing system of the invention, the aglycon is formed upondispensing and mixing of a stabilized β-glucosidase-containingcomposition and a suitable cosmetic composition containing a redanthocyanin. Due to its increased hydrophobicity, the aglycon willadhere more tightly to hydrophobic surfaces like skin and hair andtherefore is removed less efficiently by water from these surfaces.

Other enzymatic approaches are aimed at generating reactive colourantsfor oxidative colouring of hair using the dispensing system of theinvention. To this end, a stabilised laccase composition is combinedwith a suitable composition containing a colourant precursor, forexample a mono- or polyphenolic compound (see e.g. FR 2,694,018; EP504005).

The dispensing system of the invention is also advantageously used forthe in situ peroxidase-mediated formation of bactericidal compounds.Separate containment of the stabilized peroxidase on the one hand andsuitable precursor molecules, optionally plus cleaning agents, on theother hand is essential for the application of certain bactericidalagents with a limited period of bacteriological activity (cf. U.S. Pat.Nos. 4,476,108, 4,588,586). Typical examples of such naturally occurringbiocidal compounds are hypohalous acids produced by haloperoxidases fromhydrogen peroxide plus halides and hypothiocyanate produced bylactoperoxidases from hydrogen peroxide plus thiocyanate. In all cases,hydrogen peroxide is an essential but rather unstable precursor.

Hydrogen peroxide solutions can be stably incorporated in the secondaqueous composition in the dispensing system of the invention, by theuse of stabilizers such as sodium stannate or phosphonic acid (e.g.Dequest 2010). These stabilizers preferably are combined with a suitableviscosifying agent like Carbopol 934 or Rheovis CRXCA (Allied Colloids).In this approach, the first composition contains the stabilized enzymeand any hydrogen peroxide-incompatible chemicals.

The dispensing system of the invention also enables enzymatic in situgeneration of hydrogen peroxide by a hydrogen peroxide generatingenzyme, for instance an alcohol oxidase. Said hydrogen peroxidegenerating enzyme is incoporated in the same composition as theperoxidase. This form of mild disinfection, optionally combined withcleaning, is an issue not only in the field of topical application, tofight various forms of eczema or acne, but also in applications such ascontact lens cleaning and household hard surface cleaners.

The in situ generation of lipoperoxides is further example of the use ofthe dispenser of the invention. To this end, a stabilized lipoxygenasecomposition and a linoleic acid-containing composition are separatelycontained and mixed upon dispensing. It is also possible to formulatethe lipoxygenase and linoleic acid in one composition, since the highpolyol concentration used to stabilize the enzyme additionally ensuresinactivity of the enzyme. In situ generated lipoperoxides are suitablefor topical application, e.g. for dehairing or inhibition of hair growth(Puig Muset et al., Arzneimittel & Forschung, 10 (1960), 234-239).

It is also possible to combine a first composition comprising an enzymewith a second composition comprising an additional active ingredient,whereby said additional active ingredient also displays a desiredactivity in the application in question. For instance, synergy may existbetween the enzyme and the additional active ingredient.

For a totally different application than topical application, I.e. breadmaking, a dispenser is used combining baking enzymes, e.g. amylases,hemicellulases, protein disulphide isomerase, lipcxygenase and otherredox enzymes, on the one hand and additional components of a fluidbread improver on the other hand, whereby the suitable enzyme substratesare present in the dough.

An example of a combination which may create synergy is the combinationof a protease and a keratinolytic agent, such as an α-hydroxy acid. Theso-called fruitacids (α-hydroxyacids or AHA's) have emerged in thecosmetic industry as agents that can induce skin peeling and thusachieve anti-aging benefits. A disadvantage is that the low pH valuesrequired for high cell renewal rates are accompanied by irritationphenomena (see Smith, W. P., Cosmetics & Toiletries Vol 109, pp 41-48,1994). To minimize irritation, one strategy can be to lower either theAHA concentration or to increase the pH of the cosmetic composition andto compensate for the reduced skin peeling effect by adding aproteolytic enzyme to the composition.

Another example is found in the area of teeth care products. As from theintroduction of dentifrices with fluoride compounds, the incidence ofdental caries has been dramatically diminished through fluoride mediatedreinforcement of the tooth enamel layer. As a result, the bacteriumStreptococcus mutans growing in dental plaque emerges as one of the mainresidual causes for caries. Effective removal of S. mutans is onlypossible by dissolving the protective and water-insoluble polysaccharidematrix by which S. mutans adheres to the enamel (see for example Hamadaand Slade, Microbiol. Rev. 44 (1980), 331-384). As disclosed in US4,438,093, enzymes such as mutanase and dextranase prevent and suppressplaque formation. Therefore, the enzyme dispensing method of theinvention provides a convenient way to combine a fluoride-containingdentifrice with a polyol-stabilized polysaccharide degrading enzymecomposition.

The dispensing system according to the invention can be advantageouslyused in other applications than topical use. An example is in laundryhand wash applications for delicate fabrics, such as wool. In thedispensing system according to the invention, a simple liquid detergentand a stabilized enzyme composition are separately contained, to besimultaneously dispensed in the desired ratio.

Polvene Antibiotics

The development of stable aqueous compositions comprising polyenemacrolide antibiotics, such as natamycin, nystatin and amphotericin-B,always has been problematic, since these antibiotics are extremelyunstable in aqueous solutions.

For the treatment of fungal infections with natamycin, natamycincompositions with a relatively high concentration of solubilizednatamycin are required, in particular because fungi require a relativelyhigh minimal inhibitory concentration. Typically, natamycin has arelatively high solubility in organic solvents like dimethylformamide,DMSO, glycerol or propylene glycol, or in aqueous compositions at eithera low or a high pH. To obtain aqueous compositons with a highconcentration of solubilized natamycin, this antibiotic preferably issolubilized under acid or base conditions. However, the stability ofnatamycin under these conditions is rather poor. Therefore, suchnatamycin preparations are preferably made just before use.

For instance, in Dutch patent application NL 7613253, the combination ofnatamycin with citric acid is described for the treatment of horses andcows suffering from trichophyton infections, whereby the solution forthe treatment must be prepared just before use by adding an appropriateamount of water to a solid mixture of natamycin and citric acid.However, the solid mixture of natamycin and citric acid is veryhygroscopic and therefore also stable for only a rather short period.

Stable aqueous natamycin compositions are described in European PatentApplication EP 678241 which is incorporated herein by reference. Thestable natamycin compositions disclosed in EP 678241 are suspensions ofnatamycin crystals in an aqueous medium, wherein sedimentation of thecrystals is prevented by the addition of a suitable viscosifying agent.

Stable natamycin compositions, for instance those disclosed in EP 678241can be advantageously used in the dispensing system of the invention. Asimultaneous dosage of a stable natamycin suspension and a suitablebasic composition is possible. When a final formulation with a highamount of solubilized natamycin is required, said suitable basiccomposition preferably is a composition having either a low or a highpH.

Fungal skin infections in humans also form a potential target fornatamycin treatment, provided that a suitable formulation of natamycinis available. Suitable formulations of natamycin to fight theseinfections include compositions comprising a combination of natamycinwith citric acid. The use of a dispensing system according to theinvention enables the combination of a stable aqueous natamycinsuspension with a second citric acid containing composition.

Another example of a suitable basic (second) composition is ananti-dandruff shampoo.

Other Compounds

Dihydroxyacetone (DHA) is produced by fermentation and is the activeingredient of cosmetic products imparting artificial tan to the humanskin. It was recognized a long time ago that DHA is rather unstable inaqueous solution, said instability resulting in loss of skin tanningcapability as well as in the formation of skin-irritating agents such asformaldehyde and formic acid.

Stable DHA solutions are obtained by adjusting the pH of a DHA solutionto the lower ranges, preferably to a value below pH 3. As these acidicpH values are incompatible with topical use, it would be advantageous toincorporate a composition for topical use, e.g. a cosmetic composition,and the DHA-containing acidic composition in a dispensing systemaccording to the invention. The stable acidic DHA composition isneutralised upon mixing with a larger volume of a well buffered cosmeticcomposition.

Aldehyde flavour compounds are among the most unstable flavourcompounds, especially under basic conditions. As a consequence, it is amain problem how to stably incorporate flavour aldehydes, like geranial,neral and citronellal, in personal care products with a basic character,like lotions, fluid soaps and shampoos.

The dispensing system of the invention conveniently ensures a separatecontainment of a flavour-containing composition and a suitable cosmeticcomposition, thus enabling application of desirable flavour compounds inpersonal care products.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the proteolytic activity of plain and diluted stabilizedaqueous protease compositions. Proteolytic activity is measured as aclear spot on a gelatin covered film plate.

FIG. 2 shows the proteolytic activity of different proteases at a pH of4 and 7 and in the presence of α-hydroxy acids.

FIG. 3 shows a dispensing pump in which two compositions are separatelycontained. The two compositions are simultaneously dispensed and can bemixed within the dispensing pump or can be delivered separately andmixed in situ.

EXAMPLE 1 Storage Stability of Proteases in Compositions ComprisingDifferent Types of Polyols

To illustrate the storage stability of various neutral proteases indifferent types of polyols, partially purified proteases from differentsources were dissolved in liquids containing 70% (w/w) of eitherbutylene glycol, propylene glycol or PEG 6000 and stored at either 5°C., 25° C. or 40° C. At various time intervals, after dilution inaqueous buffer, remaining enzyme activities were measured.

Enzymes Used

Serine protease powder from Bacillus licheniformis was obtained fromGenencor International, Brughes, Belgium.

Neutral protease non-standardised (metallo-protease from Bacillusamyloliquefaciens) was obtained from Gist-brocades, Seclin, France.

Stabilized enzyme formulations containing either Serine protease orNeutral protease were prepared by dissolving the required quantities ofenzyme powder in either 70% (w/w) butyleneglycol (1,3-butane diol; BG),propylene glycol (1,2-propanediol; PG) or PEG 6000 (polyethylene glycol6000; PEG). To each solution, calcium acetate pH 6.0 was added to aconcentration of 0.1%, for maximal enzyme stability. Any resultingprecipitate was removed by centrifugation after which the variouslystabilized enzyme solutions were stored at either 5° C., 25° C. or 40°C. At various time intervals samples were taken and tested for residualSerine and Neutral protease activity. More than thousand-fold dilutionof the polyol containing enzyme formulations in the protease testguaranteed the absence of non-protease related interactions fromchemical contaminants.

Enzyme activities were determined following the Gist-brocades protocolfor neutral protease activity. This procedure (ISL Method Number 61195)is available from Gist-brocades Delft upon request. Briefly, theprocedure is as follows:

A strongly diluted enzyme solution is added to a solution of 0.3%Hammersten casein at 40° C., pH 7.0. After incubation during 60 minutes,protease activity is stopped by the addition of TCA. After thoroughmixing and an additional incubation at 4° C. for 30 minutes, the samplesare centrifuged. Extinction of the clear supernatant is measured at awavelength of 275 nm against distilled water. By comparison withreference protease samples, the final protease activity is obtained.

Conclusions

Serine protease dissolved in either 70% butylene glycol or propyleneglycol is more stable than neutral protease.

Butylene glycol and propylene glycol are clearly superior to PEG 6000 inenzyme stabilization.

                                      TABLE 2                                     __________________________________________________________________________    Serine protease                                                               RESIDUAL PROTEOLYTIC ACTIVITY AT:                                             5° C. 25° C.                                                                             40° C.                                        Days                                                                             BG PG  PEG                                                                              BG  PG  PEG BG  PG  PEG                                          __________________________________________________________________________    1  9670                                                                             10050                                                                             9970                                                                             9670                                                                              10050                                                                             9970                                                                              9670                                                                              10050                                                                             9970                                         9  9590                                                                             9840                                                                              9980                                                                             9770                                                                              9940                                                                              8420                                                                              9460                                                                              9580                                                                              2530                                         22 9390                                                                             9600                                                                              9500                                                                             9088                                                                              9540                                                                              4620                                                                              9120                                                                              9440                                                                              2030                                         __________________________________________________________________________

                                      TABLE 3                                     __________________________________________________________________________    Neutral Prctease                                                              RESIDUAL PROTEOLYTIC ACTIVITY AT:                                             5° C.  25° C.                                                                            40° C.                                        Days                                                                             BG  PG  PEG                                                                              BG  PG  PEG                                                                              BG  PG  PEG                                          __________________________________________________________________________    1  17900                                                                             18500                                                                             5690                                                                             17900                                                                             18500                                                                             5690                                                                             17900                                                                             18500                                                                               5690                                       9  16900                                                                             16600                                                                             3580                                                                             15900                                                                             14600                                                                             1660                                                                              3840                                                                             2540                                                                              <2000                                        22 16300                                                                             17600                                                                             3370                                                                             15200                                                                             12600                                                                             2930                                                                             <2000                                                                             4410                                                                              <2000                                        __________________________________________________________________________

EXAMPLE 2 Reactivation of Protease from a Stabilized Formulation

Materials

Neutral Protease from B. amyloliquefaciens, non-standardized, wasobtained from Gist-brocades Seclin (France).

    ______________________________________                                                          Weight (g)                                                                           End concentration                                    ______________________________________                                        Stabilized enzyme formulation                                                 Water               24.25    48.5%                                            Neutral protease    25 mg                                                     Propylene Glycol    25.0     50%                                              Hydroxypropyl Cellulose                                                                           0.75      1.5%                                            (Klucel type H from Hercules)                                                 ______________________________________                                        Preparation: after mixing the viscosifying agent hydroxypropyl                cellulose with the propylene glycol, water in which the enzyme is             dissolved is added and mixed overnight.                                       Aqueous formulation                                                           Water               48.5     97%                                              Hydroxypropyl cellulose                                                                           0.75      1.50%                                           Propylene Glycol    1.0       2%                                              ______________________________________                                         Preparation: hydroxypropyl cellulose and propylene glycol are mixed,          subsequently added to the water and mixed overnight.                     

A certain amount of the stabilized protease formulation (plain ordiluted with the aqueous formulation) is spotted on Agfapan 25 filmplate. If the enzyme is active, the gelatin on the film plate will beproteolysed, leaving a more or less clear spot. This assay mimicks theability of protease to degrade the rough top layer of keratin in vivo.

After incubation of the film plate with the plain stabilized proteaseformulation no clear spot is observed, indicating that the protease isnot active due to the high concentration of polyol. A 1:1 and a 1:2dilution of the stabilized protease formulation with the above aqueousformulation, yielding propylene glycol concentrations of 26% and 18%,respectively, results in a clear spot with a diameter increasing withthe dilution factor, indicating reactivation of the protease (see FIG.1). Although the 1:1 dilution already results in a partial reactivationof the protease, the concentration of the polyol is still too high to beacceptable for topical use.

A non-stabilized enzyme preparation, i.e. without propylene glycol,appeared to be inactive in about one week.

Thus, enzymes stabilized according to the invention are essentiallyinactive until reactivated by dilution with an appropriate composition.

EXAMPLE 3 Proteolytic Activity in Compositions Comprising Differentα-hydroxyacids

This example illustrates the efficacy of a number of protelytic enzymesunder acid conditions and in the presence of α-hydroxyacids (AHA's).Protelytic activity was assayed on photographic gelatin film.

Enzymes and Materials Used

Serine protease powder from Bacillus licheniformis was obtained fromGenencor International, Brughes, Belgium and dissolved in a mixture of35% (w/w) butylene glycol, 35% (w/w/) glycerol, 0.1% (w/w) Ca(Ac)₂ pH6.0 and water. Enzyme activity in solution was adjusted to obtain anactivity of approx. 500 Neutral Protease Units at 37° C., pH 7.0 (seeExample 1).

Aspartic proteinase liquid from Rhizomucor miehei (210-000 milk clottingunits/ml) was obtained from Gist-brocades, Seclin, France.

Cysteine proteinase powder from papaya fruits (60 million papainunits/gram, assayed according to Food Chemical Codex III) was obtainedfrom Gist-brocades, Seclin, France. Prior to use a solution of cysteineproteinase was freshly prepared by dissolving 20 mg of enzyme powder in2 ml demin. water.

AHA stock solutions were prepared according to the following protocol.Five grams of l-lactic acid (Boom Chemie, Netherlands) and five gramsglycolic acid (Merck, Germany) were each dissolved in 80 ml water,adjusted to pH 4.0 with 25% NaOH after which water was added to 100 mlto give 5% solutions of l-lactic acid and glycolic acid, respectively.Five grams of salicylic acid (Acros, Belgium) was dissolved in 80 mlwater and adjusted to pH 4.4 (dissolution at pH 4.0 was not possible)with 25% NaOH after which water was added to 100 ml to give a 5%solution of salicylic acid.

Phosphate buffer pH 7.0 and citrate HCl buffer pH 4.0 were obtained fromMerck, Germany.

Experiment

Five glass vials containing 0.1 gram serine protease solution, fiveglass vials containing 0.1 gram of aspartic proteinase and five glassvials containing 0.1 gram cysteine proteinase solution were prepared.

To each series of five vials was added 0.9 ml of respectively buffer pH7.0, buffer pH 4.0, 5% 1-lactic acid, 5% glycolic acid and 5% salicylicacid. After mixing of enzyme and buffer or acid, a 40 microliter sampleof each one of the fifteen solutions was applied in a matrix to AgfapanAPXl00 photographic film. The film with samples was then incubated for 1hour in a wetted and covered petridish at 37° C. After incubation thephotographic film was extensively rinsed with tap water and dried.Removal of the gelatin layer was used as a measure for proteolyticactivity.

From the results obtained (see FIG. 2), it appeared that serine proteasewas only active around pH 7.0 and not at pH values around 4.0, either inthe presence or absence of the various AHA's.

Whereas aspartic proteinase was not active at pH 7.0 or at pH 4.4 in thepresence of salicylic acid, the enzyme was fully active at pH 4.0 withor without 5% l-lactic acid or 5% glycolic acid.

Cysteine proteinase is active at pH 7.0 and at lower pH values in thepresence of either l-lactic, glycolic or salicylic acid. Surprisinglylow proteolytic activity was recorded at pH 4.0 in the absence of eitherone of the AHA's.

Conclusions

In combination with low pH values and AHA's, aspartic proteinases arepreferred over serine proteases.

Cysteine proteinase from papaya exhibits proteolytic activity at bothneutral and acidic pH values.

Depending on the conditions, the most appropriate proteolytic enzymeshould be selected.

EXAMPLE 4 Enzymatic Activation of Ascorbyl Phosphate

Three different enzymes with a documented fosfatase activity wereformulated in 70% propylene glycol and incubated under variousconditions with magnesium ascorbyl phosphate. Dephosphorylation ofmagnesium ascorbyl phosphate was quantitated using 600 MHz proton NMR.

Enzymes

Phytase (Aspergillus niger; without glycerol, containing approx. 12.000FTU/gram) as well as acid phosphatase (Aspergillus niger; lyophilizedpowder, containing approx. 10.000 units/gram) were obtained fromGist-brocades, Delft, the Netherlands.

Potato phosphatase (lyophilized powder) was obtained from Sigma.

Experiment

In a liquid containing 70% (w/w) propylene glycol in D₂ O and 2 mmol/lof EDTA (pH7-8), approx. 1% (w/w) of each of the above mentioned enzymepreparations was dissolved.

Ten times dilution of these enzyme stock solutions in D₂ O, containing 1(w/w) magnesium ascorbyl phosphate (NIKKO Chemicals Co, Japan), bufferedto pH 5.0 using acetic acid and kept at 37° C., resulted in a 50%degradation of magnesium ascorbyl phosphate within a period of 4 hours.

Storage of the 70% propylene glycol containing enzyme stock solution at37° C. for 1 week, did not markedly affect enzyme activity in thisassay.

Combination of enzyme and substrate in the presence of 70% (w/w)propylene glycol, clearly demonstrated inactivity of the enzyme underthese conditions. Starting from a stock solution of 5% (w/w) magnesiumascorbyl phosphate in D2O, a mixture was prepared containing:

70% (w/w) propylene glycol

1% (w/w) magnesium ascorbyl phosphate

2 mmol/l EDTA

pH 7-7.5

After complete dissolution (during which a gel-like structure wasformed), 1% (w/w) of (pre-dissolved) acid phosphatase and potatophosphatase enzyme was added. The resulting mixture was incubated for 1week at 37° C. after which the concentration of hydrolysed magnesiumascorbyl phosphate was measured versus a similar sample containing noenzyme.

As no enzymatic hydrolysis could be detected, this demonstrates oncemore the lack of enzymatic acitivity in the presence of highconcentrations of polyol.

EXAMPLE 5 Performance of Phytase Towards Magnesium Ascorbyl PhosphateHydrolysis Under Application Conditions

It is shown that phytase (NatuPhos® 5000L, Gist-brocades, Delft, theNetherlands) is effective under application conditions. In the test, 2ml of 1% magnesium ascorbyl phosphate in 100 mM Na-acetate pH 6.0 wasmixed with 200 μl enzyme solution. The enzyme solution was obtained bydiluting NatuPhos® 500OL one to five in a solution containing 70%glycerol (to obtain 377 U/g magnesium ascorbyl phosphate; 1 U is theamount of enzyme liberating 1 μmol phosphate per minute from 1% vitamineC-phosphate at pH 6.0 and 30° C.). After different incubation times, thereaction was terminated by the addition of 1 ml 20% TCA.

The conversion of magnesium ascorbyl phosphate was followed by measuringthe phosphate concentration using ³¹ P-NMR. It could be shown that at30° C. a 85% conversion of magnesium ascorbyl phosphate was obtainedwithin 30 minutes. At 37° C., a conversion of 89% was obtained in thesame period. After 60 min. incubation the yield increased up to around90%. The minimal activity needed for complete conversion within 30minutes was 122 U/g vitamin C-phosphate.

EXAMPLE 6 Enzymatic Hydrogen Peroxide Generation

Alcohol oxidase (Hansenula sp. from Sigma) powder with lowconcentrations of catalase was dissolved in water after which glycerolwas added to obtain a final glycerol concentration of 60% (w/w). Afterstorage of this stabilized enzyme solution for one month at roomtemperature, the enzyme solution was diluted ten times in an aqueoussolution containing 2% of ethanol and 0.01 M phosphate pH 7. Formationof hydrogen peroxide was demonstrated by a green-blue color developingupon immersion of a Perid teststrip (Boehringer Mannheim, Germany) intothe aqueous ethanol solution. The hydrogen peroxide thus generated cansubsequently be used as a substrate for a peroxidase.

By incorporating a hydrogen peroxide generating enzyme and a suitableperoxidase such as lactoperoxidase (Sigma) in a stabilizing liquid inthe one container and the required enzyme precursors in the othercontainer, the active biocidal compound is obtained upon mixing of thecontent of the two containers according to the method of the invention.

EXAMPLE 7 Deacetylation of Vitamin A-acetate

Enzymes

Piccantase concentrate (Rhizomucor miehei, containing approx. 30.000BGLE/g) was obtained from Gist-brocades, Seclin, France).

Maxatase pure (Bacillus subtilis, containing approx 2.16 BYU/kg) wasobtained from Genencor International B.V., Delft, Netherlands.

G999 Phospholipase L (Aspergillus niger, containing approx 000 u/g) wasobtained from Enzyme Bio-Systems Ltd., Englewood Cliffs, N.J., U.S.A.

Xylan acetylesterase (A. niger transformant TrA10 as described inEP0507369 and available through deposited microorganisms). Afterinoculation, transformant TrAlO was grown on a culture medium containing30 grams of soy pulp per liter to induce enzyme activity. After 48 hrsof growth at 30° C. under aeration and a minumum pH value of 4.0, thefermentation broth was centrifuged and the supernatant was filtered.First filtration was over Seitz K700 filters, second filtration overSeitz Supra 250 filter and germ filtration over a Seitz Supra EKS. Theresulting liquid was concentrated by a factor 10 using ulta-filtrationafter which the concentrate was lyophilized. Xylan acetylesteraseactivity in the final powder was estimated to be approx. 300 units/grampowder.

Retinol-acetate Hydrolysis

Enzyme solutions were prepared by dissolving 6 milligrams of enzymepowder (i.e. Piccantase, Maxatase and Xylan acetylesterase) in onemilliliter of demin. water. Of the liquid G999 preparation, 16microliters were diluted in one milliliter of demineralized water. Astock solution of retinol acetate (Sigma) was prepared by dissolving 55milligram retinol acetate in one milliliter of methanol.

Enzyme incubations were carried out by adding 100 microliter phosphatebuffer pH 5.5; 100 microliter of enzyme solution and 200 microliter ofretinal acetate stock solution to 500 microliter of demineralized water.After mixing, the various solutions were incubated under argon at 37° C.for either 1 or 4 hrs. Subsequently the solutions were lyophilised anddeuterated chloroform (Merck, Germany) was added. Removal of the acetatepart of retinal acetate was measured using 600 MHz proton NMR.

    ______________________________________                                                      Hydrolysis of retinol acetate                                   Enzyme          After 1 hr.                                                                             After 4 hrs.                                        ______________________________________                                        Maxatase        -         -                                                   G999            -         +                                                   Xylan acetylesterase                                                                          0         ++                                                  Piccantase      +         ++                                                  ______________________________________                                         - = no hydrolysis; 0 = detectible hydrolysis; + = significant hydrolysis;     ++ = complete hydrolysis                                                 

Lipid Hydrolysis

In some applications of esterified vitamins, reactivation of thevitamins by enzymes with lipolytic activities is less desirable. Forexample during reactivation of vitamin precursors included in cosmetics,enzymic degradation of the oils included in the cosmetic product shouldbe avoided. In this respect it would be advantageous to avail of vitaminactivating enzymes with no degradative effect towards triglyceride oils.To establish the triglyceride degrading effect of the retinal acetatedegrading enzymes mentioned above, the three active enzymes weresubjected to a test in which hydrolysis of emulsified olive oil wasquantitated.

An emulsion of olive oil in polyvinyl alcohol and water was prepared.Oil droplets larger than 10 microns do not occur. After addition of theenzym solution, the drop of pH created by the enzymatic liberation offatty acids is compensated by a constant titration with sodiumhydroxide. After a fixed incubation period at pH 7.5 and 37° C., thetotal quantity of sodium hydroxide used is measured and used as a(relative) value for lipolytic activity. The details of this method havebeen described in document CQA 4047 and can be obtained fromGist-brocades, Seclin, France, upon request.

    ______________________________________                                                        Lipolytic activity/gram                                       Enzyme          enzyme                                                        ______________________________________                                        G999            <1                                                            Xylan acetylesterase                                                                          110                                                           Piccantase concentrate                                                                        20.000                                                        ______________________________________                                    

Conclusion

Various enzymes including lipases are able to deacetylate vitaminA-acetate.

Both G999 and Xylan acetylesterase are of particular interest becausethese enzymes combine deacetylation activity with very low lipolyticactivity. Moreover, the natural substrates for these enzymes (i.e.lysophospholipids and acetylated xylans) do not normally occur on thehuman skin.

EXAMPLE 8 Suspension of Crystalline Protease

In multiple dose dispensing systems, proper suspension of theimmobilized and stabilized material is important to guarantee an evendosing of the active material into the final composition. The suspensionmethod should enable long term storage without sedimentation of theimmobilized active material.

Materials

ChiroCLEC-BL, an aqueous solution of cross-linked protease (subtilisin)crystals, obtained from Altrus Biologics Inc., Cambridge, Mass, USA.

Carbopol-Ultrez-10, a viscosifying polymer, was obtained from BFGoodrich, The Hague, Netherlands.

From a homogeneous aqueous suspension of ChiroCLEC-BL, a sample of 4.8grams was obtained and centrifuged to collect the crystalline material.After removal of the supernatant, the crystals were washed once indemineralized water and recentrifuged, after which the crystals weresuspended in a final amount of 10 grams of a composition containing 35%glycerol, 35% butylene glycol and 0.4% Carbopol. The pH of the resultingsuspension was adjusted to 5.5 using triethanolamine.

After thorough homogenisation the suspension was placed at 40° C. Nosedimentation of the crystals was observed after a one month incubationat this temperature. Final dilution of the enzyme suspension restoresenzymatic activity.

EXAMPLE 9 Stabilization of Dihydroxvacetone

The stability of DHA solutions was analyzed at different temperaturesand pH values and with different antioxidant additions.

NMR Analysis

Identification as well as quantification of DHA degradation products wasperformed on a Bruker AMX-600 spectrometer, operating at a ¹ H frequencyof 600 Mhz. A 5 mm inverse probe was used. The huge water signal wassuppressed by means of simple presaturation (2 s) or by means ofpresaturation with composite pulses (2 s). The delay of 2 s does notallow for complete relaxation between observe pulses, and thereforequantitative results should be considered as semi-quantitative, i.e. asystematic error may occur of up to 100%. However, the results withinone series of measurements can be used for a meaningful comparison.

HPLC Analysis

Some samples were analysed by means of HPLC. To 2 ml of each sample 400μl 20% perchloric acid was added. The following equipment and conditionswere employed:

    ______________________________________                                        HPLC pump      Varian LC 5010                                                 Injection volume                                                                             20 μl                                                       Detectors      IOTA differential refractometer                                               Varian UV-5, 215 nm                                            Column         Aminex Hpx 87 H 300 × 7.8 mm                             Eluens         0.01 N H.sub.2 SO.sub.4                                        Flow           0.6 ml/min                                                     ______________________________________                                    

This method was only used to quantify DHA in the experiments of series1.

DHA Degradation in Water of 50° C.

Solutions were prepared of 5% DHA in a buffer solution of either 0.1 Mphosphate (pH 7) or 0.1 M pyrophosphate (pH 5.5 and pH 8.5) in D₂ 0. Insome cases ascorbic acid was added. Samples were incubated in a stove at50° C. for up to 7 days.

DHA degradation 50° C. in water, sample conditions and residual DHAafter 2, 5 and 7 days:

    ______________________________________                                                         DHA (g/l) after days of                                      Ascorbic acid    incubation        Final                                      pH   g/l        0       2    5      7    pH                                   ______________________________________                                        5.5  --         49      31   22     17   4.9                                  7.0  --         49      18   13     11   5.6                                  8.5  --         46      2.4  1.1    0.8  7.5                                  7.0  9          48      18   13     11   5.7                                  5.7  9          47      28   19     14   5.3                                  ______________________________________                                    

Conclusions

Acidic pH conditions improve the storage stability of DHA.

The presence of a reducing agent like ascorbic acid has no significanteffect on the storage stability of DHA.

DHA Degrdation at 40° C. Under Various Conditions

Solutions were prepared of 5% DHA in a buffer solution of 0.2 mpyrophosphate. To all samples 10% of D₂ O was added, containingt-butanol, which served as an internal standard. The final concentrationof t-butanol was 0.45 mg/ml. Samples nr. 4 and 5 were prepared in amixture of H₂ O/glycol =1/1. Moreover, 10% t-butanol was added to thesesamples and in the case of sample nr. 5 also 15 mg (dl) α-tocoferol.Samples were flushed with N₂ gas or air for 30 minutes. These conditionsare also summarised underneath. Samples were incubated at 40° C. for 35days.

Sample composition and treatment:

    ______________________________________                                        Sample nr DHA    pH      flush additions                                      ______________________________________                                        1         5      6       air   --                                             2         5      6       N.sub.2                                                                             --                                             3         5      5       air   --                                             4         5      5       N.sub.2                                                                             glycol/H.sub.2 O/t-bu 45/45/10                 5         5      5       N.sub.2                                                                             see 4 + 0.3 tocoferol                          6         5      2.6     air   --                                             ______________________________________                                    

DHA degradation after 35 days at 40° C.:

    ______________________________________                                                Remaining DHA                                                                             ppm formic acid                                                                            ppm acetic acid                              Sample nr.                                                                            initial = 100%                                                                            formed       formed                                       ______________________________________                                        1       20%         1600         2400                                         2       20%         1400         2600                                         3       40%         1000         1200                                         4       30%         nd           nd                                           5       30%         nd           nd                                           6       90%         200          300                                          ______________________________________                                         na: not determined                                                       

Conclusions:

Significant stabilisation of DHA can only be obtained by adjusting thepH to very low values.

EXAMPLE 10

Natamycin in Shampoo

In this example the use of a dispensing system is described which isdesigned to simultaneously deliver a natamycin containing compositionand a shampoo composition in a ratio of 1:10.

11 g of natamycin trihydrate, 1 g of xanthan gum (Keltrol® RD, KelcoInternational Limited), 8 g of lactose, 0.5 g of citric acid and 0.055 gof sodiumcitrate dihydrate were mixed together in a Turbula® mixer. Thetotal mixture was then suspended into 480 ml of water. The resultingsuspension had a pH of 4.8 and contained 2% of pure natamycin.

25 ml of the above suspension was put into one compartment of thedispenser, while the other compartment was filled with 225 ml of ashampoo composition.

At the application, a shampoo dosage containing 2000 ppm of natamycinwas obtained.

EXAMPLE 11 Natamycin for Trichophyton

110 g of natamycin trihydrate and 1 g of xanthan gum were suspended in388 ml of distilled water and then sterilized by heat treatment. The pHof the suspension was 6.5. After standing for at least 4 weeks nosedimentation was observed. HPLC analysis showed that the natamycincontent immediately after the preparation and after 4 weeks storing atambient temperature was respectively 20.1 and 20.2% (w/w).

200 g citric acid monohydrate and 4 g of xanthan gum were dissolved in876 ml of distilled water and sterilized by heat treatment. The pH ofthe solution was 3.0.

By using a dispensing system which is designed to simultaneously deliverthe natamycin composition and the citric acid solution in a ratio of1:9, each dosage contains about 2% of natamycin. To obtain a solutioncontaining 200 or 100 ppm of natamycin for the treatment of the animals,the dosage may be simply diluted 100 to 200 times by mixing with water.Thus a dispenser containing 250 g of the sample (25 g of the natamycinsuspension and 225 g of the citric acid solution) will be sufficient formaking an overall quantity of 25 to 50 1 of the treatment solution.

By using a dispensing system which is able to simultaneously deliver astable natamycin-containing suspension and a citric acid composition,both compositions being separately contained, a system is obtained whichconveniently enables the cost-effective preparation of small dosages.

EXAMPLE 12 Dispensing Systems

Dispensing systems suited for simultaneously dosing two separatelycontained incompatible compounds, are well known. As such, thedispensing system schematically depicted in FIG. 3 (dispenser fromMaplast, Italy) is just one example out of a number of products whichrange from small, two-chambered single use pouches to tubes usingdifferent product compartments or tubes compartmentalized usingextrudable, viscous and relatively inert materials to separate theincompatible compounds.

The dispenser shown in FIG. 3 is able to simultaneously dose twocompounds separately contained in A and B by pressing dosing head C.Pressing dosing head C activates two small pumps which subsequentlydispense the two compounds in approximately equal volumes. Depending onthe design of the dosing head, the compounds can be dosed in twoseparate streams or in just one stream.

According to the present invention, a dispensing unit is required thatis able to deliver a stabilized, aqueous enzyme composition togetherwith a non-enzyme containing basic composition in a ratio of forinstance 1:2.

Translated to the dispenser depicted in FIG. 2, this would mean that oneof the two pumps is able to dose at least twice the volume of the otherpump in just one stroke of dosing head C.

Translated to a two-chambered single use pouch, this would mean that thechamber containing the enzyme composition contains at least half as muchproduct volume as the other chamber.

Translated to a two-compartment tube, this would mean that under equalpressure the discharge orifice for the compartment containing thecomposition without enzyme allows the passage of at least twice as muchproduct as the discharge orifice of the other compartment.

Translated to a tube which is compartmentalized using extrudablematerial, this would mean that the composition without enzyme is presentinside the tube in at least double the volume of the enzyme-containingproduct.

What is claimed is:
 1. A dual chamber dispensing system for thesimultaneous dosing of two aqueous compositions comprising first andsecond separate chambers that respectively contain a first aqueouscomposition in said first chamber and a second aqueous composition insaid second chamber and dispensing means that cause the simultaneousdosing of said two aqueous compositions to obtain a final compositionthat is directly applied; wherein,said first composition is a stableformulation of an enzyme in the presence of a polyol; said secondcomposition is an aqueous composition, which reactivates the enzyme;said final composition contains an effective concentration of saidreactivated enzyme; and said first composition and said secondcomposition are dispensed in a ratio from about 1:1 to about 1:50. 2.The dispensing system of claim 1, wherein said means dispenses saidfirst composition and said second composition in a ratio from about 1:2to about 1:20.
 3. The dispensing system of claim 2, wherein said meansdispenses said first composition and said second composition aredispensed in a ratio from about 1:5 to about 1:10.
 4. The dispensingsystem of claim 1, wherein said second composition contains anadditional active ingredient.
 5. The dispensing system of claim 1,wherein said first composition additionally contains a viscosifyingagent.
 6. The dispensing system of claim 1, wherein said biologicallyeffective compound is formulated in a particle form.
 7. The dispensingsystem of claim 6, wherein said first composition additionally containsa viscosifying agent which forms a three-dimensional network in anaqueous composition.
 8. The dispensing system of claim 6, wherein saidparticle form is a crystal.
 9. The dispensing system of claim 8, whereinsaid first composition additionally contains a viscosifying agent whichforms a three-dimensional network in an aqueous composition.
 10. Thedispensing system of claim 1, wherein said enzyme is formulated in aparticle form.
 11. The dispensing system of claim 10, wherein saidparticle form is obtained by immobilizing said enzyme on a solidcarrier.
 12. The dispensing system of claim 10, wherein said particleform is obtained by crystallization of said enzyme.
 13. The dispensingsystem of claim 10, wherein said first composition additionally containsa viscosifying agent which forms a three-dimensional network in anaqueous composition.
 14. The dispensing system of claim 1, wherein saidsecond composition contains an active compound precursor which is asubstrate for said enzyme.
 15. The dispensing system of claim 14,wherein said precursor is a vitamin precursor.
 16. The dispensing systemof claim 1, wherein said polyol is present in said first composition ina concentration of about 20-90%.
 17. The dispensing system of claim 16,wherein said polyol is present in said first composition in aconcentration of about 30-90%.
 18. The dispensing system of claim 17,wherein said polyol is present in said first composition in aconcentration of about 40-90%.
 19. The dispensing system of claim 18,wherein said polyol is present in said first composition in aconcentration of about 50-90%.
 20. The dispensing system of claim 19,wherein said polyol is present in said first composition in aconcentration of about 60-80%.
 21. The dispensing system of claim 1,wherein said enzyme is a protease.
 22. The dispensing system of claim 1,wherein said enzyme is an esterase having a preference for lower chainacyl groups.
 23. The dispensing system of claim 22, wherein saidesterase has a preference for lower chain acyl groups having 1 to 9carbon atoms.
 24. The dispensing system of claim 1, wherein said enzymeis a phosphatase.
 25. The dispensing system of claim 24, wherein saidphosphatase is a phytase.
 26. A method for topical application of abiologically effective compound comprising topically administering to asubject said enzyme contained in a carrier by simultaneous dosing twoaqueous compositions from the dispensing system of claim 1.